Protein conjugates, i.e. proteins conjugated to a molecule of interest via a linker, are known in the art. For example, fluorescent labeling is a powerful technique for in vitro and in vivo visualisation, covalent immobilization of proteins is a useful strategy for industrial application and PEGylation of proteins leads to significantly enhanced circulation time. In addition, there is great interest in antibody-conjugates wherein the molecule of interest is a drug, for example a cytotoxic chemical. Antibody-drug-conjugates are known in the art, and consist of a recombinant antibody covalently bound to a cytotoxic chemical via a synthetic linker.
Protein conjugates known from the prior art are commonly prepared by conjugation of a functional group to the side chain of amino acid lysine or cysteine, by acylation or alkylation, respectively.
For lysines, conjugation takes place preferentially at lysine side chains with highest steric accessibility, the lowest pKa, or a combination thereof. Disadvantage of this method is that site-control of conjugation is low.
Better control of site-specificity is obtained by alkylation of cysteines, based on the fact that typically no or few free cysteines are present in a typical protein, thereby offering the option of alkylating only those cysteines that are already present in reduced form or selectively engineered into a protein. Alternatively, cysteines can be selectively liberated by a (partial) reductive step. For example, selective cysteine liberation by reduction is typically performed by treatment of a protein with a reducing agent (e.g. tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT)), leading to conversion of a disulfide bond into two free thiols. The liberated thiols are then alkylated with an electrophilic reagent, typically based on a maleimide chemistry, which generally proceeds fast and with high selectivity, or with haloacetamides, which also show strong preference for cysteine but side-reactions with lysine side-chains may be encountered.
One recent report (N. M. Okeley et al., Bioconj. Chem. 2013, 24, 1650, incorporated by reference herein) describes the metabolic incorporation of 6-thiofucose into the glycan of a monoclonal antibody, followed by reduction-oxidation, then maleimide conjugation. Interestingly, it was found that the 6-thiofucose maleimide conjugate described above was found to display enhanced stability with respect to cysteine maleimide conjugates. However, efficiency of incorporation of 6-thiofucose was found to be only 70%.
An alternative variant of maleimide conjugation, which was applied for the generation of an antibody-drug conjugate, involves a strategy where not the nucleophilic thiol is introduced in the monoclonal antibody, but rather the maleimide. For example, T-DM1 is prepared by first (random) conjugation of lysines with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), thereby effectively charging the antibody with maleimides. In the next stage of the process, the maleimide-functionalized antibody is treated with thiol-functionalized maytansinoid, leading to the conjugate. Hence, this is an example where the antibody is converted into an electrophilic reaction partner (instead of the common use of nucleophilic amino acid side chains for conjugation), upon treatment with SMCC. However, also in this case, by nature of the approach, only random conjugation of antibody is achieved.
Notwithstanding the versatility of the above technologies, a general disadvantage of protein conjugates obtained via alkylation with maleimides is that in general the resulting conjugates can be unstable due to the reverse of alkylation, i.e. a retro-Michael reaction.
An alternative strategy to prepare conjugates of a glycoprotein, a subclass of all proteins, involves the selective attachment of functional moieties to one (or more) of the glycans present on the glycoprotein.
One example of conjugation to glycoproteins involves the generation of one or more aldehyde functions on the protein's glycan structure, either by chemical means (sodium periodate) or by enzymatic means (galactose oxidase). The latter aldehyde function can subsequently be employed for a selective conjugation process, for example by condensation with a functionalized hydroxylamine or hydrazine molecule, thereby generating an oxime-linked or hydrazone-linked protein conjugate, respectively. However, it is known that oximes and hydrazones, in particular derived from aliphatic aldehydes, also show limited stability over time in water or at lower pH. For example, gemtuzumab ozogamicin is an oxime-linked antibody-drug conjugate and is known to suffer from premature deconjugation in vivo.
Another example of glycoprotein conjugation involves the use of a glycosyltransferase for controlled modification of the glycan with a monosaccharide of choice.
Qasba et al. disclose in WO 2004/063344 and in J. Biol. Chem. 2002, 277, 20833, both incorporated by reference herein, that mutant galactosyltransferases GalT(Y289L), GalT(Y289I) and GalT(Y289N) can enzymatically attach GalNAc to a non-reducing GlcNAc sugar ((β-benzyl-GlcNAc).
WO 2007/095506 and WO 2008/029281 (Invitrogen Corporation), incorporated by reference herein, disclose that the combination of GalT(Y289L) mutant with C2-substituted azidoacetamido-galactose UDP-derivative (UDP-GalNAz) leads to the incorporation of GalNAz at a terminal non-reducing GlcNAc of a glycan. Subsequent conjugation by Staudinger ligation or with copper-catalyzed click chemistry then provides the respective antibody conjugates wherein a fluorescent alkyne probe is conjugated to an antibody. WO 2007/095506 and WO 2008/029281 further disclose that trimming of the glycan can take place with endo H, thereby hydrolyzing a GlcNAc-GlcNAc glycosidic bond and liberating a GlcNAc for enzymatic introduction of GalNAz.
A disadvantage of the latter approach is the removal of most of the hydrophilic sugars, which may not only hamper conjugation because of the single sugar remaining in the linker, but may also increase protein aggregation due to decreased hydrophilicity of the linker connecting the protein and the functional molecule, in particular when the functional molecule is hydrophobic. It is desirable in such case to prepare protein conjugates with linkers that are both longer (more sugars) and more hydrophilic (better water-solubility).
Qasba et al. disclose in Bioconjugate Chem. 2009, 20, 1228, incorporated by reference herein, that β-galactosidase-treated monoclonal antibodies (e.g. Rituxan, Remicade, Herceptin) having a G0 glycoform (obtained by treatment of the crude mAbs with galactosidase) are fully regalactosylated to the G2 glycoform after transfer of GalNAz to the terminal GlcNAc residues of the glycan, leading to tetraazido-substituted antibodies, i.e. two GalNAz moieties per heavy chain. The transfer of a galactose moiety comprising a C2-substituted keto group (C2-keto-Gal) to the terminal GlcNAc residues of a G0 glycoform glycan, as well as the linking of C2-keto-Gal to aminooxy biotin, is also disclosed.
Based on the above, it is clear that galactose can be introduced to proteins featuring a terminal GlcNAc-moiety upon treatment with wild type Gal-T1/UDP-Gal (leading to Gal-GlcNAc-protein), while N-acetylgalactosamine can be introduced upon treatment with GalT1 mutant Y289L (affording GalNAc-GlcNAc-protein). It has also been shown by Elling et al. (Chem Bio Chem 2001, 2, 884, incorporated by reference herein) that a variety of human galactosyltransferases (β4-Gal-T1, β4-Gal-T4 and (β3-Gal-T5), but not bovine β4-Gal-T1, can accommodate a 6-biotinylated modification of galactose in UDP-Gal, in the absence of Mn2+, leading to effective transfer to model proteins BSA-(GlcNAc)17 and ovalbumin. Similarly, Pannecoucke et al. (Tetrahedron Lett. 2008, 49, 2294, incorporated by reference herein) demonstrated that commercially available bovine β4-Gal-T1 under standard conditions is also able to transfer UDP-6-azidogalactose to a model GlcNAc-substrate, but the transfer to a GlcNAc-protein was not demonstrated.
In WO 2007/133855 (University of Maryland Biotechnology Institute), incorporated by reference herein, a chemoenzymatic method for the preparation of a homogeneous glycoprotein or glycopeptide is disclosed, involving a two-stage strategy entailing first trimming of the near-complete glycan tree (under the action of endo A, endo H or endo S) leaving only the core N-acetylglucosamine (GlcNAc) moiety (the so-called GlcNAc-protein), followed by a reglycosylation event wherein, in the presence of a catalyst comprising a mutant endoglycosidase (ENGase), an oligosaccharide moiety is transferred to the GlcNAc-protein to yield a homogeneous glycoprotein or glycopeptide. A strategy for azide-functionalized glycoproteins is disclosed, wherein a GlcNAc-protein is reacted in the presence of ENGase with a tetrasaccharide oxazoline containing two 6-azidomannose moieties, thereby introducing two azides simultaneously in the glycan. The azide-functionalized glycoprotein may then be catalytically reacted in a “click chemistry” cycloaddition reaction, in the presence of a catalyst (e.g. a Cu(I) catalyst) with a terminal alkyne bearing a functional moiety X of interest. No actual examples of said click chemistry are disclosed.
In J. Am. Chem. Soc. 2012, 134, 8030, incorporated by reference herein, Davis et al. disclose the transfer of oligosaccharide oxazolines on a core-fucosylated as well as nonfucosylated core-GlcNAc-Fc domain of intact antibodies, in the presence of glycosynthase EndoS.
In J. Am. Chem. Soc. 2012, 134, 12308, incorporated by reference herein, Wang et al. disclose the transfer of a tetrasaccharide oxazoline containing two 6-azidomannose moieties on core-fucosylated as well as nonfucosylated core-GlcNAc-Fc domain of intact antibodies (Rituximab) in the presence of glycosynthase mutants EndoS-D233A and EndoS-D233Q.
However, a disadvantage of the glycosynthase strategies disclosed in WO 2007/133855, J. Am. Chem. Soc. 2012, 134, 8030 and J. Am. Chem. Soc. 2012, 134, 12308 is the lengthy and complex synthesis of the required azido-containing oligosaccharide oxazolines. In addition, the azido-containing oligosaccharide oxazolines comprise two azido groups. To date, it has not been shown whether this process may be suitable for the introduction of only one azido group on an antibody glycan.